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CYP1B1 Augments the Mesenchymal, Claudin-Low, and Chemoresistant Phenotypes of Triple-Negative Breast Cancer Cells

Cytochrome P4501B1 (CYP1B1) is elevated in breast cancer. Studies indicate a relationship between CYP1B1 and aggressive cancer phenotypes. Here, we report on in vitro studies in triple-negative breast cancer cell lines, where knockdown (KD) of CYP1B1 was used to determine the influence of its expression on invasive cell phenotypes. CYP1B1 KD in MDA-MB-231 cells resulted in the loss of mesenchymal morphology, altered expression of epithelial–mesenchymal genes, and increased claudin (CLDN) RNA and protein. CYP1B1 KD cells had increased cell-to-cell contact and paracellular barrier function, a reduced rate of cell proliferation, abrogation of migratory and invasive activity, and diminished spheroid formation. Analysis of clinical breast cancer tumor samples revealed an association between tumors exhibiting higher CYP1B1 RNA levels and diminished overall and disease-free survival. Tumor expression of CYP1B1 was inversely associated with CLDN7 expression, and CYP1B1HI/CLDN7LOW identified patients with lower median survival. Cells with CYP1B1 KD had an enhanced chemosensitivity to paclitaxel, 5-fluorouracil, and cisplatin. Our findings that CYP1B1 KD can increase chemosensitivity points to therapeutic targeting of this enzyme. CYP1B1 inhibitors in combination with chemotherapeutic drugs may provide a novel targeted and effective approach to adjuvant or neoadjuvant therapy against certain forms of highly metastatic breast cancer.

Introduction
Cytochrome P450 1B1 (CYP1B1) belongs to the superfamily of mixed function oxidases involved in the cell detoxification and metabolic activation of many structurally diverse compounds [1,2]. CYP1B1 is a major extrahepatic enzyme involved in estrogen and xenobiotic metabolism [2,3]. In humans and mice, mutations of CYP1B1 lead to abnormal development of the trabecular meshwork and increased intraocular pressure that result in primary congenital glaucoma [4].
Breast cancer is a heterogeneous disease that is divided into subtypes based on the expression of the estrogen receptor (ER), progesterone receptor (PR), and amplification of the human epidermal growth factor receptor (HER2). Traditionally, five breast cancer intrinsic subtypes were commonly described including, normal breast-like, basal-like (BL), HER2-enriched (HER2 + ), luminal A (LumA) and luminal B (LumB) [5]. Each intrinsic subtype is its own distinct disease, displaying significant disparities in rate of incidence, therapeutic response, and survival [5]. Such classification has aided the development of targeted therapies for patients expressing ER, PR, or HER2. Moreover, further subclassification, for example low-risk and high-risk LumA, has identified cohorts of patients more or less likely to benefit from specific treatment regimens [6]. In the case of patients with tumors lacking the expression of these receptors few targeted treatments are available. BL breast cancers, with minimal or undetectable ER and PR expression and are negative for HER2 amplification, are collectively termed triple-negative breast cancer (TNBC). TNBC tends to form large tumors that have a high rate of axillary lymph node involvement, and is more aggressive, accounting for 10-20% of invasive breast cancers [7,8]. These patients have a poor prognosis with low five-year and overall survival rates. Recently, an additional breast cancer subtype was identified by microarray gene expression analysis of breast tumors from humans and mice [9]. Named the Claudin (CLDN)-low (CL) subtype [9], CL tumors are mostly negative for ER, PR, and HER2 expression [10], and have diminished expression of epithelial tight junction, and cell-to-cell adhesion proteins including CLDN3, CLDN4, and CLDN7. CL tumors express markers associated with epithelial-mesenchymal transition (EMT) such as vimentin (VIM), and transcriptional repressors of cadherin 1 (CDH1) including twist family BHLH transcription factor 1 (TWIST1), twist family BHLH transcription factor 2 (TWIST2), zinc finger E-box binding homeobox 1 (ZEB1), zinc finger E-box binding homeobox 2 (ZEB2), snail family transcriptional repressor 1 (SNAI1) and snail family transcriptional repressor 2 (SNAI2) [5,8]. The identification and expression of features commonly associated with TNBC tumors suggest that the CL subtype is a subgroup of TNBC [11]. However, recent studies demonstrate that other intrinsic subtypes of breast cancer may express the claudin-low phenotype [12]. These reports indicate that CL may be a phenotype of multiple intrinsic breast tumor subtypes, albeit having a higher prevalence in the BL subtype [13,14].
Elevated levels of CYP1B1 are reported in many types of cancer. Breast cancer CYP1B1 elevation is reported for RNA and protein [15], suggesting that CYP1B1 may play a role in the initiation and/or progression of this cancer. Early studies of breast cancer cell lines report that aryl hydrocarbon receptor (AHR)-mediated induction of CYP1B1 is higher in cells having a mesenchymal morphology. Such ER-, invasive and tumorigenic cells lines have higher levels of CYP1B1 catalyzed estrogen 4-hydroxylase activity [16]. Two of these cell lines used here, the MDA-MB-231 and MDA-MB-157 lines, are classified by genome-wide transcript stratification as triple negative basal cells having a mesenchymal phenotype [17]. Subsequent studies further identify these cell lines as being of the CL subtype [5].
The functional significance of CYP1B1 expression in cancer was first demonstrated in endometrial cancers [18]. Immunohistochemical analysis demonstrate higher levels of CYP1B1 in endometrial adenocarcinoma relative to adjacent normal tissues. CYP1B1 protein is higher in undifferentiated and tumorigenic endometrial carcinoma cell lines. Depletion of CYP1B1 by small interfering RNA (siRNA) in these cells decreases cell proliferation, induces cell cycle arrest, increases apoptosis, and decreases Matrigel cell invasion [18]. To test the hypothesis that suppression of CYP1B1 diminishes the aggressive characteristics of TNBC cells, we used short hairpin RNA (shRNA) to lower CYP1B1 in MDA-MB-231 cells; to replicate certain findings, we used siRNA knockdown (KD) in MDA-MB-231 and MDA-MD-157 cells. The results of these studies indicate that CYP1B1 expression augments multiple phenotypes associated with aggressive characteristics of TNBC, and that elevated expression in breast tumors is associated with poor prognosis. Our findings that CYP1B1 KD can increase chemosensitivity supports the idea that therapeutic targeting of this enzyme, in combination with chemotherapeutic drugs, may provide a novel targeted approach to adjuvant or neoadjuvant therapy against certain forms of highly metastatic breast cancer.

CYP1B1 Knockdown Decreases Mesenchymal Phenotypes in Triple-Negative Breast Cancer Cell Lines
MDA-MB-231 cells were transduced with control vector (CV) or one of three CYP1B1 shRNA-expressing lentiviruses. CYP1B1 shRNA (62323 or 62324) transduced cells displayed some cells with a rounded cell morphology. CYP1B1 shRNA (62326) transduced cells showed a marked change in cell morphology, growing as a monolayer with rounded shape and increased cell-to-cell contact ( Figure 1A). An initial screen demonstrated that the CYP1B1 shRNA transduced cells decreased CYP1B1 protein by 48% (62323), 33% (62324) and 76% (62326) compared to the CV. Based on these results, the CV and 62326 cell lines were selected for further study. The stability of cell line 62326 was checked after multiple passages. Relative to CYP1B1 levels in CV cells, CYP1B1 RNA remained significantly decreased by 68% in 62326 cells ( Figure 1B, left); protein remained significantly decreased by 80% ( Figure 1B, right). Analysis of a select subset of mesenchymal regulators and markers showed aspects of mesenchymal-epithelial transition (MET), consistent with the changes in mesenchymal phenotypes. However, while certain regulators of the mesenchymal phenotypes, namely SNAI2, ZEB1 and ZEB2 were strongly decreased by CYP1B1 KD, SNAI1 was elevated 3-fold. Moreover, VIM and FN1 were not significantly decreased ( Figure 1C). Parallel measures of epithelial cell markers showed similar results. KRT8, KRT18 and LAMA1 were each significantly increased, while CDH1 was not ( Figure 1D). Levels of CDH3 were significantly elevated by nearly 3-fold ( Figure 1E) suggesting that these CYP1B1 KD cells may be in an intermediate state [19], with transition between mesenchymal and epithelial characteristics, termed epithelial-mesenchymal plasticity [20,21]. The CYP1B1 KD cells showed a markedly slower rate of proliferation in comparison to CV transduced cells ( Figure 1F). This phenotype was replicated by siRNA KD of CYP1B1 in both MDA-MB-231 and MDA-MB-157 cells. The siRNA transfection of these cells did not decrease viability at 24 h. (Supplementary Figure S1A-C). Furthermore, the siRNA KD cells had a much slower growth rate. Levels of caspase 3 were modestly (60%), yet significantly elevated. Caspase 3 cleavage was also increased about 67%. However, no cleavage of poly(ADP-ribose) polymerase (PARP) protein was detected (Supplementary Figure S1D). Thus, it does not appear that apoptosis accounts for all the observed differences in cell proliferation rates.   Consistent with the altered mesenchymal to epithelial morphology ( Figures 1A and S1B), CYP1B1 KD resulted in a robust and significant decrease in cell migration ( Figures 1G and S1E), and the effects to decrease invasion through Matrigel were even greater ( Figures 1H and S1F). Multiple spheroid formation in Matrigel suspension was much greater in CV cells than in CYP1B1 KD cells ( Figure 1I), consistent with lowered growth rates and loss of invasive phenotypes. Collectively, these results indicate an important role of CYP1B1 in the highly invasive phenotypes of TNBC cells.

CYP1B1 Suppression Is Associated with Increased CLDN Expression in CL TNBC Cell Lines
As high CYP1B1 activity is associated with mesenchymal TNBC cells lines [16] having a CL phenotype [17], we explored the relationship between CYP1B1 suppression and CLDN expression. Levels of CLDN1 RNA and protein were strongly elevated in CYP1B1 KD cells ( Figure S2B). Immunoblot of CLDN7 protein revealed a 9-fold increase in this protein in CYP1B1 KD MDA-MB-231 cells ( Figures 2B and S2D). Similar increases of CLDN1 and CLDN7 proteins were observed by indirect immunofluorescence in comparisons of CV and CYP1B1KD MDA-MB-231 cells. Representative confocal microscope images showed increased CLDN1 and CLDN7 immunofluorescence staining around the perimeter of the cells ( Figures 2C and S2C,D). These increases corresponded with the change of cell morphology from mesenchymal to epithelial (Supplementary Figure S2C,D). Both CLDN1 and CLDN7 proteins were organized at the apical-lateral borders at points of cell-to-cell contact in CYP1B1 suppressed cells in comparison to the CV cells ( Figure 2C). Furthermore, dual detection of CLDN7 and tight junction protein 1 (TJP1), a scaffolding protein essential for proper tight junction formation [22], indicated that both proteins organized at the apical-lateral borders of the KD cells, especially at areas of cell-to-cell contact ( Figure 2D). Co-localization of these proteins was determined by correlation analyses. The Pearson correlation coefficient was 0.71; the Mander's overlap coefficients were c1 = 0.977 and c2 = 0.992. Both approaches were supportive of co-localization. To determine whether these observed increases provided the cells with improved tight junction barrier function, we measured paracellular permeability. At both 96 h and 120 h post plating, CYP1B1 KD cells showed significant decreases of paracellular flux in comparison to CV cells ( Figure 2E). Together, these data revealed that CYP1B1 KD is associated with increased CLDN levels in CL-low TNBC cell lines, resulting in increased adhesion and tight junction function.

Association of RNA Levels of CYP1B1 and CLDN7 in Clinical Data of Breast Cancer Samples
Microarray gene expression data with corresponding clinical data of breast cancer samples were accessed from the UNC Microarray Database, UNC337 [5]. Breast cancer samples were divided into tertiles by CYP1B1 RNA levels: low, medium, and high. Samples with the highest CYP1B1 RNA levels were compared to samples with the lowest CYP1B1 RNA levels. Kaplan-Meier analysis demonstrated that breast cancer patients with high CYP1B1 expression had decreased probability of overall survival ( Figure 3A) and disease-free survival ( Figure 3B) than those with low CYP1B1 expression. Further analysis considered the expression of CYP1B1 within five intrinsic subtypes: BL, CL, HER2 + , LumA, and LumB [5,8]. CYP1B1 expression was significantly higher in CL samples, and significantly lower in LumB samples ( Figure 3C). Linear regression analysis identified a significant negative regression coefficient representing CYP1B1 and CLDN7 RNA levels ( Figure 3D). Grouped by CYP1B1 HI /CLDN7 LOW , 61% of CL and BL samples contained more CYP1B1 HI /CLDN7 LOW samples while 27% of all other samples were CYP1B1 HI /CLDN7 LOW ( Figure 3E). Comparison of CYP1B1 HI /CLDN7 LOW CL and BL samples to all other samples revealed a significant decrease in the median survival and disease-free survival of patients identified as CYP1B1 HI /CLDN7 LOW ( Figure 3F). Overall, these analyses of this set of clinical data showed a negative correlation of expression of CYP1B1 and CLDN7 in breast cancer, consistent with our findings in CL TNBC ( Figure 2). Moreover, these results indicate that patients with CYP1B1 HI /CLDN7 LOW tumors may have a poorer prognosis.  Figure 4C). Because cell proliferation reflects both the rates of cell replication and cell death, and because CYP1B1 KD had such a large effect on the rate of cell replication ( Figure 1F), we determined cell viability 72 h post treatment with a single effective concentration of each agent. In untreated cells, the percentage of non-viable cells was less than 8%, with no significant difference between CV and CYP1B1 KD cells ( Figure 4D-F). Treatment of cells with 1 µM PTX significantly increased the percentage of non-viable cells from 13% in CV cells to 40% in CYP1B1 KD cells ( Figure 4D). In contrast, treatment with 10 µM 5-FU resulted in no change in the percentage of nonviable cells in either CV or CYP1B1 KD cells ( Figure 4E). Treatment of cells with 100 µM CPT significantly increased the percentage of non-viable cells from 50% in CV cells to 82% in CYP1B1 KD cells ( Figure 4F). Preliminary studies of the highly potent and selective CYP1B1 inhibitor 2,3 ,4,5 -tetramethoxystilbene (TMS) [23] revealed that TMS decreased cell invasion in a concentration-dependent manner. Moreover, co-treatment of TMS and 5-FU showed a significantly greater decrease in cell proliferation than did 5-FU alone (Supplementary Figure S4A,B). within the box. The IQR is 25th percentile (Quartile 1) to 75th percentile (Quartile 3). The notch shows the 95% confidence interval of the median, and the whiskers include values within ±1.5 IQR and 99.3% of the data points. Two-sample t-test (p ≤ 0.05) of the means were corrected for multiple comparisons using Bonferroni's correction. (D) CYP1B1 RNA levels predict CLDN7 RNA levels The coefficient of the linear regression line, Beta one hat (̂1), is the estimate of the slope of the line that best fits the data points. The significance of this value was determined by t-test. (E) Combined BL and CL tumor samples contain more CYP1B1 HI /CLDN7 LOW samples than combined (LumA+ B+ HER2 + ) samples. Samples with >10% increase in CYP1B1 RNA levels and >10% decrease in CLDN7 RNA levels were classified as CYP1B1 HI /CLDN7 LOW relative to RNA levels in all other samples.

Discussion
CYP1B1 KD in TNBC cells induced changes from mesenchymal to epithelial morphology. This was accompanied by decreased expression of known regulators of the mesenchymal phenotype and increased expression of epithelial markers. Of importance, robust changes in cell proliferation, migration, and invasion were observed. Multiple spheroid formation was markedly and significantly decreased in the CYP1B1 KD cells. proliferation reflects both the rates of cell replication and cell death, and because CYP1B1 KD had such a large effect on the rate of cell replication ( Figure 1F), we determined cell viability 72 h post treatment with a single effective concentration of each agent. In Values are the mean ± SD, n = 5-9. The * indicates a significant difference (p ≤ 0.05) in comparison to CV within the same concentration of drug and the † indicates a significant difference from vehicle within the same the cell line using two-way ANOVA followed by Tukey's multiple comparisons.

Discussion
CYP1B1 KD in TNBC cells induced changes from mesenchymal to epithelial morphology. This was accompanied by decreased expression of known regulators of the mesenchymal phenotype and increased expression of epithelial markers. Of importance, robust changes in cell proliferation, migration, and invasion were observed. Multiple spheroid formation was markedly and significantly decreased in the CYP1B1 KD cells. proliferation reflects both the rates of cell replication and cell death, and because CYP1B1 KD had such a large effect on the rate of cell replication ( Figure 1F), we determined cell viability 72 h post treatment with a single effective concentration of each agent. In untreated cells, the percentage of non-viable cells was less than 8%, with no significant difference between CV and CYP1B1 KD cells ( Figure 4D-F). Treatment of cells with 1 µM PTX significantly increased the percentage of non-viable cells from 13% in CV cells to 40% in CYP1B1 KD cells ( Figure 4D). In contrast, treatment with 10 µM 5-FU resulted in no change in the percentage non-viable cells in either CV or CYP1B1 KD cells ( Figure 4E). Treatment of cells with 100 µM CPT significantly increased the percentage of non-viable cells from 50% in CV cells to 82% in CYP1B1 KD cells ( Figure 4F). Preliminary studies of the highly potent and selective CYP1B1 inhibitor 2,3 ,4,5 -tetramethoxystilbene (TMS) [23] revealed that TMS decreased cell invasion in a concentration-dependent manner. Moreover, co-treatment of TMS and 5-FU showed a significantly greater decrease in cell proliferation that did 5-FU alone (Supplementary Figure S4A,B). Collectively, these results indicate that CYP1B1 KD results in MET, characterized as a population of cells in which the migratory and spindle-shaped mesenchymal cells transition to a more organized and polarized epithelial cell morphology, with less aggressive characteristics [24].
In a study of inflammatory breast cancer (IBC) clinical data, CYP1B1 and AHR RNA were positively correlated and elevated in IBC samples. Expression levels of each positively correlated with expression of WNT5A/B and β-catenin [25], known downstream effectors of mammary tumors induced by AHR ligand activation [26]. Expression of WNT5A/B and β-catenin were correlated with markers of the cancer stem cell (CSC) phenotype, and AHR-antagonist treatment of MDA-MB-231 or SUM149 breast cancer cells resulted in significantly lower levels of WNT5A RNA. Sherr and colleagues [25,[27][28][29] developed a model of TNBC where AHR activation drives properties associated with tumor progression including migration, invasion, expression of CSC markers, and CSC characteristics of enhanced spheroid formation, tumor formation in NOD/SCID mice, and chemoresistance. In this model, the AHR increases expression of indoleamine 2,3 dioxygenase (IDO)/tryptophan dioxygenase (TDO) enzymes to establish an IDO/TDO-AHR regulatory loop [27]. In this feed-forward signaling loop, IDO/TDO-catalyzed metabolism of tryptophan produces kynurenine, an AHR ligand, which acts to increase tumor aggression. This loop is autoregulated by AHR-dependent elevation of the AHR repressor, and CYP1A1 and CYP1B1 enzymes that metabolize kynurenine ligands to decrease their bioavailability [29], thus forming a cancer progression circuit where AHR-regulated CYP1B1 levels correlate with AHR activity. In this model, expression of CYP1B1 is viewed as a surrogate or biomarker of AHR signaling and hence the activity of this cancer progression circuit [25,29].
This model predicts that increases of CYP1B1 would increase AHR ligand metabolism and decrease AHR activity and aggressive phenotypes, while decreases in CYP1B1 would increase ligand availability, AHR activity, and aggressive phenotypes. Two studies contradict this model and indicate that CYP1B1 expression may play a significant role in tumor progression. In a study by Kwon et al. [30], ectopic expression of CYP1B1 in either ER-MCF-10F or ER+ MCF7 cell lines drove increased expression and nuclear localization of β-catenin, mesenchymal morphology, EMT-associated gene expression, and invasion. MCF-10F cells, while immortal, are neither transformed nor tumorigenic. Thus, while the establishment of the IDO/TDO tumor progression circuit seems unlikely, the overexpression of CYP1B1 nonetheless drove EMT in these cells [30]. Moreover, overexpression of CYP1B1 is not known to activate the AHR, and treatment of breast cancer cell lines with 4-hydroxyestradiol, the primary estrogen product of CYP1B1, results in effects similar to ectopic CYP1B1 expression [30]. Plausible targets of estrogen metabolites include ERβ and G protein estrogen receptor (GPER) [31]. Interestingly, GPER is reported to regulate CYP1B1 expression in ER-breast cancer and modulation of both GPER and CYP1B1 alter tumor cell growth in vitro and in vivo [32]. Thus, regulation of CYP1B1 by pathways in addition to the AHR may be important determinants to its expression and roles in tumor progression. The second line of evidence indicating that CYP1B1 contributes to these aggressive phenotypes is provided by the results of this current study where depletion of CYP1B1 blocked migration, invasion, and EMT. Collectively, these results indicate that CYP1B1 deserves additional study as an independent driver of aggressive tumor characteristics in TNBC.
Loss of tight junction proteins and lack of cell-to-cell contact are characteristics of multiple carcinomas, including TNBC. These features are viewed as essential for cellular invasion and migration that is necessary for metastasis [33]. CLDN proteins create tight junctions between cells, regulating paracellular permeability. Because MDA-MB-231 and MDA-MB-157 are characterized as claudin low TNBC cell lines and CYP1B1 KD cells displayed epithelial-like cell morphology and high frequency of cell-to-cell contacts, we measured the expression of CLDN RNAs and proteins. In CYP1B1KD cells, RNA levels of CLDN1, CLDN3, CLDN4 and CLDN 7 were elevated, as were proteins for CLDN1 and CLDN7. Confocal microscopy revealed organized cells with both CLDN1 and TJP1 expressed at the apical lateral borders. Moreover, functional tight junctions were demonstrated by decreases of paracellular permeability. Collectively, these data indicate that CYP1B1 contributes to the CL phenotype observed in the same TNBC cell lines.
To explore the possible clinical significance of CYP1B1 expression in breast cancer and CL breast cancer, we analyzed the UNC337 Microarray Database [5]. Analysis of the 311 tumor samples showed significant differences in overall and disease-free survival when the samples were grouped by tertiles into either high versus low expression of CYP1B1. Analysis of CYP1B1 expression by previously assigned intrinsic breast cancer subtypes [5] showed significantly higher expression in the CL subtype and significantly lower expression in the luminal B subtype. Analysis of all tumor samples showed a significant negative regression coefficient for CYP1B1 and CLDN7 expression values, consistent with our cell-based experiments. Sorting all samples by CYP1B1 HI /CLDN7 LOW transcript levels revealed that 61% of the samples meeting this criterion were independently classified as either CL or BL. This finding is consistent with recent studies investigating CL as a complex phenotype rather than a novel intrinsic breast cancer subtype [13,14]. Overall, the CL breast tumors appear to reflect the characteristics of their cell of origin [14], and stratification of CL status does not indicate a poorer survival within an intrinsic subtype [13]. Fougner and colleagues [13] report a much greater proportion of CL tumors in the BL (14.6%), than in the HER2 + (0.9%), LumA (1.3%) or LumB (0.6%) intrinsic subtypes.
Despite such controversies related to CLDN expression and function in tumorigenesis, several studies indicate that increased CLDN1, CLDN4, or CLDN7 can act as a suppressor of metastasis, and a potential inducer of MET in breast, lung, colon, pancreatic, gastric epithelial and nasopharyngeal carcinoma cells [24,[34][35][36][37]. Similarly, CLDN16 overexpression in MDA-MB-231 cells results in an epithelial-like cell shape, increased frequency of cell-to-cell contact, and attenuation of aggressive phenotypes [38]. Conversely, reduced expression of CLDN1 and CLDN7 correlates with invasion and metastases in gastric epithelial cells and carcinoma of the esophagus, respectively [39,40]. In the current study, both overall and disease-free survival were significantly lower in the samples classified as CYP1B1 HI /CLDN7 LOW . Thus, this simple two-factor classification (CYP1B1 HI /CLDN7 LOW ) may be sufficient to identify breast cancer patients with poorer prognostic outcomes and aid in the development of novel targeted treatments for these aggressive cancers.
TNBC has a poor prognosis, in part, relating to its aggressive phenotypes and resistance to chemotherapy and radiation [41]. Here we showed that suppression of CYP1B1 results in dose-dependent decreases in the rate of proliferation of MDA-MB-231 cells to multiple chemotherapeutic agents including PTX, 5-FU and CPT relative to control cells. Further, these CYP1B1-drug interactions displayed differing effects on cell viability. For PTX and CPT, CYP1B1 KD enhanced chemical-dependent cytotoxicity, while for the antimetabolite 5-FU, CYP1B1 KD interacted to affect cell proliferation, but not cell viability. Thus, CYP1B1 modulation may affect multiple mechanisms that provide chemotherapeutic benefit in breast cancer treatment. Chemical inhibitors of CYP1B1 are reported to increase the sensitivity of TNBC cell lines to CPT [42] and other compounds in several tumor types [43].
Such inhibition is also reported to impart cardioprotective effects [43] indicating that there may be added benefits to chemotherapy approaches that include CYP1B1 inhibitors.
The mechanisms of chemoresistance are complex [44,45]. In some cases, CYP1B1 may act to alter chemotherapeutic disposition and thereby drug efficacy [46]. However, the data presented here and by others [25,30,32] support the idea that CYP1B1 directly contributes to complex cancer phenotypes including chemoresistance that may not directly involve drug metabolism. For example, the potent and selective CYP1B1 inhibitor, TMS, decreases cell growth and migration, as well as tumor growth in xenograft models in vivo [30,32,47]. Consistent with these reports, our studies of TMS revealed dose-dependent decreases in cell invasion and cell proliferation. Cells co-treated with 5-FU and TMS showed further decreases in cell proliferation. Overall, elevated expression of CYP1B1 in TNBC cells appears to enhance mesenchymal and claudin-low characteristics while knockdown or inhibition of this enzyme weakens their associated invasive and chemoresistant properties. These observations point towards novel therapeutic strategies for certain subtypes of breast cancer.
The results of the current study, while compelling, are limited in that they rely on the manipulation of CYP1B1 levels in a subset of breast cancer cell lines. These findings should be strengthened and expanded by future studies using patient derived cancer cells, as well as in vivo experimental studies of tumor growth and metastases, and patient-derived xenograft models. Such studies are necessary for a thorough preclinical evaluation of the importance of CYP1B1 to the aggressive phenotypes associated with TNBC.

CYP1B1 shRNA Transduction Cell Lines
MDA-MB-231 cells were transduced with five different lentiviral constructs containing shRNA specific for CYP1B1. To obtain stable CYP1B1 suppressed cells, MDA-MB-231 cells were seeded in a 12-well plate at a density of 4 × 10 4 cells/well and incubated overnight. Medium was then replaced with medium containing 5 µg/mL polybrene and the cells were transduced with a multiplicity of infection of 2 transducing units. 48 h post-transduction, medium was replaced with medium containing 2 µg/mL puromycin. The following CYP1B1 MISSION shRNA Lentiviral Transduction Particles constructs (Sigma Millipore) were tested: TRC numbers are 62323, 62324, 62325, 62326, and 62327, or empty vector backbone (CV, SHC001V).

Quantitative PCR (qPCR)
Total RNA was isolated from cells using STAT-60 (CS502, Tel-Test, Friendswood, TX, USA). qPCR was performed as described [48] using the following conditions: denaturation at 95 • C for 15 min, followed by denaturation at 95 • C for 30 s with 40 cycles, annealing from 52-58 • C for 30 s (dependent on primer pair) and extension at 72 • C for 30 s. Gene expression was normalized to ACTB expression. Primer sets used in this study are provided in Supplementary Table S1.

Cell Proliferation Assays
Cell proliferation was assessed using the Fluoreporter Blue Fluorometric dsDNA quantitation method (F2962, Thermo Fisher Scientific) or MTT assay (CT01, Sigma Millipore). For the Fluoreporter assay, cell number was measured by using the Hoescht 33258 according to the manufacturer's procedure. Fluorescence was measured at excitation of 360 nm and emission of 460 nm using a Synergy H1 Microplate Reader and Gen 5 software version 2.09 (BioTek, Winooski, VT, USA). For the MTT assay, the manufacturer's instructions were followed.

Invasion and Migration Assay
The invasion and migration assays followed [49] with minor modifications. The invasion assay was performed using 24-well polyethylene terephthalate membrane transwell inserts with 8.0 µm pores (353493, Corning, Corning, NY, USA). Inserts were coated with Matrigel (1 mg/mL; 356237, Corning) and allowed to polymerize overnight at 37 • C. Matrigel was rehydrated for 2 h before addition of cells to the insert. Cells were seeded at 1 × 10 5 cells/cm 2 in phenol-red free, serum free DMEM in the upper chamber of the Matrigel coated insert. FBS (10%) was added to the lower chamber, basal side of the membrane. Inserts were incubated at 37 • C and cells were allowed to invade for 22 h. The non-invading cells on the apical side of the membrane were removed with a cotton swab. Cells that invaded through the Matrigel and attached to the basal side of the membrane were fixed in 100% methanol and stained with 0.05% crystal violet for 10 min and rinsed two times in distilled water. The inserts were visualized using an EVOS digital inverted microscope (Thermo Fisher Scientific) with 200× magnification. Images of 16 fields per insert were taken. Experiments were performed with five replicate inserts. The invaded cells on the basal side of the insert were counted manually and shown as the mean of 80 fields per treatment. The cell migration assay was performed identical to the invasion assay, excluding the use of Matrigel coating of transwell inserts.

Morphology Assessment
Cells grown in culture were visualized using an EVOS Digital Inverted Microscope with 200× magnification for morphology assessment. Images of 10 fields per well were taken at random and counted for mesenchymal or epithelial morphology.

Trypan Blue Exclusion Assay
Cells collected by trypsinization were added to media containing 10% FBS. After centrifugation, the pellets were resuspended in 0.4% trypan blue (T8154, Sigma): PBS (1:1) immediately before four quadrants of cells were counted manually using a hemocytometer under an inverted phase contrast Zeiss Axiovert 100 microscope at 10× magnification. Non-viable cells (blue cells) and the total number of cells (live and dead) were counted in each quadrant before percent non-viable cells was calculated.

Multiple Spheroid Formation Assay
Cells (5000) were seeded in triplicate on Matrigel-coated (1 mg/mL; 356237, Corning) chamber slides in serum free medium, 2% suspension of Matrigel as described [51], and grown for up to 25 days. Images of 10 individual fields were captured for each of three chambers with the EVOS Digital Inverted Microscope at 100× magnification. Colonies (>50 cells) were counted manually.

Statistical Analysis of UNC337 Patient Tumor Sample Data
For all analyses, the normal/normal-like samples were removed from the dataset, leaving 311 samples: CL (n = 37), BL (n = 73), HER2 + (n = 39), LumA (n = 100), and LumB (n = 62), GSE 18229 [5]. These 311 samples were separated into tertiles by CYP1B1 RNA levels (low n = 103, medium n = 104, and high n = 104). Log-rank analyses of the CYP1B1-dependent differences in survival and disease-free survival were assessed using the Mantel-Cox test of the Kaplan-Meier curves generated in survival: Survival Analysis package in R * [https://cran.r-project.org] accessed on 5 February 2019. For each subtype, median log 2 expression values were plotted as box and whisker plots relative to overall mean CYP1B1 expression level. Pairwise comparisons of the group mean values were performed using a t-test with Bonferroni's correction for multiple testing. For analyses of CYP1B1 and CLDN7 expression, data are reported as a log 2 (fold-change) relative to the mean expression value for all samples. Linear regression analysis was performed in R using CYP1B1 and CLDN7 data from all 311 samples. The relationship between levels of CYP1B1 and CLDN7 was estimated from the regression coefficient and tested by t-test. The CYP1B1 HI /CLDN7 LOW population was defined as samples with >10% increase in CYP1B1 expression relative to the mean for all samples, and >10% decrease in CLDN7 expression relative to the mean for all samples. Significance of differences in median survival and disease-free survival between CYP1B1 HI /CLDN7 LOW samples and all other samples was assessed using a non-parametric Mann-Whitney test due to non-normal distribution of survival data values.

Statistical Analysis
All experiments were performed with cells at different passage numbers and repeated at least two times. Major findings were replicated using two techniques for CYP1B1 KD (shRNA and siRNA) and certain results were replicated in multiple cell lines. Results reported for analyses of a single clinical breast data set are supported by functional studies performed in multiple cell lines. Statistical analysis was performed using GraphPad Prism software (version 7.03; GraphPad Software, La Jolla, CA, USA) and R * [https: //cran.r-project.org] accessed on 5 February 2019. A two-tailed Student's t-test was used to compare results from the control vector samples to the CYP1B1 shRNA transduced samples. Where appropriate, two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests were used to compare the effects of CYP1B1 expression levels on treatments. One-way ANOVA was used to compare results from the mock, non-specific, and CYP1B1 siRNA samples. Data were considered statistically significant with a p value of ≤ 0.05. Details of the methods used for clinical sample analysis are provided in that specific section and the figure legend.

Conclusions
In our studies of claudin-low TNBC cell lines, CYP1B1 appears to augment mesenchymal and CL phenotypes. Furthermore, knockdown of CYP1B1 lessens invasion, spheroid formation, and chemoresistance. These results indicate a need to better understand the function of CYP1B1 in the context of TNBC and the significance of the CYP1B1 HI /CLDN7 LOW classification of breast tumors. Collectively, the present study has implications for the potential combined use of a CYP1B1 inhibitor with chemotherapy for both adjuvant and neoadjuvant treatment approaches to TNBC.

Informed Consent Statement:
The human tumor data analyzed were accessed from GSE 18229, kindly made available by Prat and colleagues. As these data are de-identified and publicly available, this does not constitute human subject research. As such, ethics approval and consent are not applicable.

Data Availability Statement:
The human tumor data were accessed from GSE 18229, kindly made available by Prat and colleagues [5]. The CYP1B1 antibody [50] is available from the corresponding author.


s2fieldsofstudy:
Biology
Medicine
Engineering
Materials Science

text:
Synthesis, characterization, and cytotoxicity of doxorubicin-loaded polycaprolactone nanocapsules as controlled anti-hepatocellular carcinoma drug release system

Background Free doxorubicin (Dox) is used as a chemotherapeutic agent against hepatocellular carcinoma (HCC), but it results in cardiotoxicty as a major side effect. Hence, a controlled Dox drug delivery system is extremely demanded. Methods Dox was loaded into the non-toxic biodegradable polycaprolactone (PCL) nanocapsules using the double emulsion method. Characterization of Dox-PCL nanocapsules was done using transmission electron microscopy and dynamic light scattering. Encapsulation efficiency and drug loading capacity were quantified using UV–visible spectrophotometry. Drug release was investigated in vitro at both normal (7.4) and cancer (4.8) pHs. Cytotoxicity of Dox-PCL nanocapsules against free Dox was evaluated using the MTT test on normal (Vero) and hepatic cancer (HepG2) cell lines. Results Spherical nanocapsules (212 ± 2 nm) were succeffully prepared with a zeta potential of (-22.3 ± 2 mv) and a polydisperse index of (0.019 ± 0.01) with a narrow size distribution pattern. The encapsulation efficiency was (73.15 ± 4%) with a drug loading capacity of (16.88 ± 2%). Importantlly, Dox-release from nanocapsules was faster at cancer pH (98%) than at physiological pH (26%). Moreover, although Dox-PCL nanocapsules were less toxic on the normal cell line (GI 50 = 17.99 ± 8.62 µg/ml) than free Dox (GI 50 = 16.53 ± 1.06 µg/ml), the encapsulated Dox showed higher toxic effect on cancer HepG2 cells compared to that caused by the free drug (GI 50 = 2.46 ± 0.49 and 4.22 ± 0.04 µg/ml, respectively). Conclusion The constructed Dox-PCL nanocapsules constitute a potentially controlled anti-HCC therapy with minimal systemic exposure. Graphical Abstract

Background Globally, liver cancer is considered as the second cause of death with an annual incidence of ~ 850,000 cases [1]. In Egypt, liver cancer is considered as the most common type of cancer in males and the second most common type of cancer in females [2,3].
Hepatocellular carcinoma (HCC) represents 90% of all primary liver cases. Worldwide, HCC represents the seventh most common cancer in females and the fifth in males [4]. In Egypt, liver cancer constitutes 1.68% of total cancer cases and HCC accounts for 70.48% of all liver tumors [5].
Cirrhosis is caused by many factors such as infection with hepatitis B or hepatitis C viruses, alcoholism, inherited metabolic diseases, diabetes, smoking as well as exposure to aflatoxins: a group of mycotoxins produced by the Aspergillus fungus in foodstuffs like corn and peanuts during storage in warm damp conditions [6].
HCC treatment requires careful screening and selection of therapies such as radiotherapy, chemotherapy, and surgery. Although radio-and chemotherapeutic drugs are capable of shrinking tumor growth for a short time, they require multiple schedules of treatment. In addition, chemotherapeutic drugs do not only kill cancerous cells but also the healthy normal ones, resulting in significant toxic side effects for the patients [7].
Examples of these cytotoxic chemotherapeutic drugs are doxorubicin (Dox), mitomycin C, cisplatin, and mitoxantrone [8]. Dox (C 27 H 30 ClNO 11 ) is an anthracycline antibiotic, which is widely used for the treatment of many types of tumors such as acute leukaemia, liver, lung, breast, ovarian, stomach, uterine, testicular and bladder cancers [9].
Several mechanisms have been proposed and subjected to controversy. One of the most popular explanations is the ability of Dox to inhibit DNA synthesis through intercalating with the DNA topoisomerase II enzyme [10][11][12]. Dox inhibition of these enzymes causes failure of resolving the knotting and supercoiling of the DNA double strands during transcription, recombination, and cellular replication, which results in the death of the cancer cell or any other cell by inducing cell apoptosis [13].
However, many adverse events are caused by the administration of Dox including; anaemia, vomiting, skin pigmentation, diarrhea, dehydration, gastrointestinal tract bleeding, hyperuricemia and cardiotoxicity [14]. Cardiotoxicity is considered as the main obstacle of treatment using Dox [15]. The first case of cardiotoxicity caused by the repeated treatment of doxorubicin was presented as heart failure in 1967 [16]. The cardiotoxicity induced by Dox occurs through the accumulation of both cellular reactive oxygen species (ROS) and mitochondrial irons, as a result of the binding of Dox molecules to the cell membranes [17].
Mitochondria are then the most affected intracellular organelles following cellular exposure to Dox, which is accumulated in the mitochondrial membrane mainly due to its high afinity to bind cardiolipin, a mitochondrial membrane phospholipid [18][19][20][21]. Cardiolipin is responsible for maintaining the structure, function, cardiac energy metabolism of the mitochondria and cell survival [22,23]. Binding of cardiolipin to Dox results in the bloking of the mitochondrial electron transport chain through the deactivation of complexes I, III, and IV [24,25]. Moreover, superoxide anion (O 2 − ) is produced upon the reoxidation of these species via transferring one electron to molecular oxygen (O 2 ). Furthermore, hydrogen peroxide (H 2 O 2 ) is synthesized by manganese superoxide dismutase, and can undergo subsequent Fe or Cu mediated conversions into more reactive hydroxyl radicals [26,27]. Consequently, these reactive species can cause lipid peroxidation as well as nucleic acid and protein oxidation. Hence, serious destructive effects in mitochnodria are resulted including damage of mitochondrial DNA (mtDNA), decrease of ATP concentrations, cardiolipin peroxidation, and alteration in the permeability of the inner mitochnondrial membrane [28][29][30].
In addition, tumor cells exhibited multidrug resistance (MDR), attributed to the presence of P-glycoprotein (P-gp), which can pump Dox out, resulting in reducing its intracellular accumulation and therapeutic efficacy. These drawbacks of Dox efficiency on cancer therapy resulted in the restriction of its clinical applications and the need to develop new drug formulations [18].
The advances in nanotechnology and nanomedicine have led to the use of nanocarriers as drug delivery systems to deliver a certain chemotherapeutic drug to cancer cells [19]. This is actually the main reason why much effort has been dedicated to the development of pharmaceutical and colloidal forms encapsulating this drug, to reduce its undesirable side effects [22].
The ideal drug delivery system (DDS) should have the ability to deliver the appropriate therapeutic drug dose to the target cells and release it in a controlled manner in the human body in order to maintain its applicable concentration for a certain period of time. This helps to diminish the side effects of drugs and maximize their therapeutic efficacy [24].
Among a range of drug nanoparticles that are synthesized using polymers are Polymeric Nanocapsules, which have a central core surrounded by a polymeric outer shell. These are mostly applicable as nanocarriers to deliver anticancer drugs. Moreover, they are able to incorporate higher doses of chemotherapeutic drugs, which results in the enhancement of their therapeutic effects [26,28].
Recently, many scientists prepared different forms of Dox-loaded polymeric nanocapsules, using various polymers to minimize its toxic side effects [29].
In this study, polycaprolactone (PCL) was chosen to produce polymeric nanocapsules, as it is considered as an interesting biocompatible and biodegradable synthetic polymer for the preparation of nanocarriers with potential therapeutic applications. PCL is approved by the United States Food and Drug Administration (FDA) and commonly used to encapsulate a wide range of anticancer drugs [32].
The aim of the present study was the synthesis of polymeric nanocapsules with an aqueous inner core enclosed by an organic layer and both are surrounded by an outer aqueous shell. This double-layer structure can readily guarantee the incorporation of the anticancer Dox drug into the nanocapsules cores, whereas the hydrophilic outer shell can provide stabilization for the nanocapsules without the need for additional surfactant. The prepared Dox polymeric nanocapsules are to be highly promising for safe chemotherapeutic applications by reducing its cytotoxicity against normal Vero cell line, and increasing its therapeutic effect against cancer HepG2 cell line.

Preparation of Dox-PCL nanocapsules
Dox-PCL nanocapsules were produced by modified double emulsion technique (W/O/W) as described by [33]. Briefly, 1.5 ml Dox solution (2 mg/ml) were emulsified with 10 mg PCL dissolved in 8 ml DCM using a high speed homogenizer (Tekmar, UK) for 3 min at 5000 rpm to create the first emulsion phase of water-in-oil (W1/O). Then, the first emulsion is transferred to an aqueous solution containing 35 ml 2% PVA and 5 ml 0.5% PEG and are homogenized for 5 min at 8000 rpm to form the second emulsion phase (W1/O/W2). The resulting mixture was left stirred on a magnetic stirrer (C-MAG HS 7, IKA, China) overnight at room temperature, in the dark. After evaporation of DCM, the remaining solution was centrifuged using an ultracentrifuge (supra25K, Hanil science industrial, Korea) for 1 h at 13,000 rpm and 10 • C. Finally, the supernatant was decanted and stored to be used later on ("Evaluation of Dox encapsulation efficiency and drug loading" section) and the pellet was resuspended in 2 ml deionized H 2 O and directly fed into the freeze dryer (Edwards Modulyo, UK) to produce a dried powder of Dox-PCL nanocapsules, which was then collected and kept at 4 °C.

Evaluation of Dox encapsulation efficiency and drug loading
The encapsulation efficiency percentage (EE %) and drug loading content (DL%) of Dox-PCL nanocapsules were indirectly measured using the supernatant resulted from the previous step ("Preparation of Dox-PCL nanocapsules" section), i.e., after centrifugation of the final nanoemulsion mixture solution produced from the final phase of the double emulsion procedure used to prepare Dox-PCL nanocapsules at 13,000 rpm for 1 h. The sample was measured in triplicate using UV-Vis spectrophotometer (Model: se6100 UV-Vis double beam, Abbota Corporation, USA) at 480 nm and the results were presented as the mean ± SD. Standard calibration curve of known Dox.HCl concentrations (1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0156, 0.0078, 0.0039, 0.00195, 0.000975 and 0.0000487) mg/ml was constructed [34]. EE % and DL % were calculated using the following Eq. (A.1) and Eq. (A.2): where, M of Dox and M of polymers are the initial mass of Dox and PCL, respectively, used in the double emulsion technique.

In vitro release study of Dox from PCL nanocapsules
The released amount of Dox from the polymeric nanocapsules was investigated at two pH values in simulated dissolution media: pH 4.8 simulating the pH of cancerous cells, and pH 7.4 simulating the environment of the normal tissues, over a period of 23 days according to [35,36]. Briefly, 1-mg sample of Dox-PCL nanocapsules was added to a 5-ml volume of 1X PBS at (pH 7.4) or (pH 4.8)

Morphology of Dox-PCL nanocapsules
The shape of the nanocapsules was determined using a transmission electron microscope (TEM) (JEM-1400, Jeol, USA). A small drop of the nanocapsules suspension was added to the carbon-coated grid, stained with uranyl acetate, and air-dried before measurement.

Assessment of nanocapsule size and size distribution
The average size and PDI of the nanocapsules were measured using DLS (Malvern, UK). The sample was diluted and sonicated for 5 min and then measured at room temperature.

Zeta potential analysis of the nanocapsules
The surface charges of the obtained nanocapsules were measured as a function of zeta potential by DLS (Nanotrac, wave2, UK).

In vitro studies of Dox-PCL nanocapsules on the viability of normal and cancer cell lines
Normal Vero cell line and liver cancer HepG2 cell line were chosen to evaluate the cytotoxicity of both free Dox and Dox-PCL nanocapsules by MTT assay. The cells were cultured according to the protocol proposed by American Type Culture Collection (ATCC) [37]. HepG2 and Vero cells were cultured separately in 6-well cell culture plates at a density of 30000 cells per well for 24 h at 37 °C in a 5% CO 2 incubator. Based on the study [38], various concentration ranges of the free drug, Dox, have been prepared (42.2 to 0 µg/ml). Equivalent amounts of encapsulated drug inside drug-containing PCL nanocapsules (250 to 0 µg/ml) were adjusted to the same concentrations of the free drug by the corresponding cell culture The cumulative drug release(%) = amount of Dox released initial amount of Dox × 100 medium. The MTT assay was done based on the calculation of the drug loading capacity of anticancer drug, Dox, inside PCL nanocapsules: Each 1 mg of the nanocapsules contains 0.1688 mg/ml of Dox. For example, the first concentration of Dox-PCL nanocapsules 250 μg/ml contains 42.2 μg/ml of the free Dox as shown in Table 1.
Briefly, cells were then treated for 72 h with Dox-PCL nanocapsules solution at concentrations of (250, 125, 62.5, 31.25, 15.6, 7.8, 3.9, 2, 1 and 0) µg/ml. Free Dox drug was used as a positive control at concentrations of (42.2, 21.1, 11, 5.3, 2.6, 1.3, 0.65, 0.325, 0) µg/ml. Then, 20 µl of MTT reagent (5 mg/ml) was added to each well and the plates were incubated for 3 h at 37 °C in a 5% CO 2 incubator. Finally, the media with MTT solution was carefully aspirated and replaced with 150 µl DMSO by a multichannel micropipette, the plates were covered with aluminum foil, and agitated on an orbital shaker for 15 min. Absorbance at λ 570 nm was measured using a microplate reader. The cell viability is expressed as a percentage of viability using Eq. (C.1): where, A Treated is the average absorbance in wells containing cells treated with a defined concentration of free Dox or Dox-PCL nanocapsules, A Blank is the absorbance of blank (DMSO), and A control is the absorbance of the untreated cells that were used as negative control [39,40].
In parallel, the cytotoxic effect of empty PCL nanocapsules (0 to 250 µg/ml) was also studied as a blank control.
For inhibitory concentration (GI 50) calculation of free Dox and Dox-loaded PCL, an excel sheet was used after calculating the inhibition percentage at various concentrations of both Dox forms and the results are expressed as mean ± SD.

Statistical analysis
The data were presented as the mean ± SD of at least three replicates. The test of significance was performed by GraphPad Prism 7 (San Diego, California, USA). Twotailed multiple T-test was employed to determine the significance of differences between normal and cancer cells. The ρ-value < 0.05 was considered to indicate a statistically significant difference.e.

Calculation of EE % and DL % of Dox-PCL nanocapsules
Firstly, a calibration curve of known drug concentrations vs absorbance at λ 480 nm was constructed as shown in Fig. 1. Dox-PCL nanocapsules were successfully prepared by the double emulsion technique. Encapsulation efficiency (EE %) and drug loading (DL %) were 73.15% ± 4 and 16.88 ± 2%, respectively.

Characterization of Dox-PCL nanocapsules
A TEM image revealed that Dox-PCL nanocapsules prepared by double emulsion were spherical as shown in Fig. 2. The nanocapsules appeared as bright spherical entities surrounded by a dark stain. It was apparent that Dox was assembled in the nanocapsule core surrounded by the hydrophobic PCL part and the aqueous phase of PVA was exposed to the outer shell. The particle   Fig. 3. The average size of nanocapsules determined by DLS was 212 nm ± 2, the zeta potential was -22.3 ± 2 mv, and PDI was 0.019 ± 0.01 with a narrow monodispersed unimodal size distribution pattern.

Cumulative release of Dox from PCL nanocapsules at normal and cancer pHs
Two calibration curves of known drug concentrations vs absorbance at λ 480 nm were constructed at 2different pHs (normal 7.4; cancer 4.8) as shown in Figs. 4 and 5.
The profile of Dox-release indicated only one phase of release as shown in Fig. 6. Approximately 97.45% of Dox was released within 72 h at pH 4.8 (cancer pH). Whereas, the rate of Dox release at pH 7.4 (normal pH) was approximately 99.67% over 23 days, which is notably slower than that at the cancer pH.

In vitro cytotoxicity evaluation of free Dox, blank PCL and Dox-PCL nanocapsules by MTT assay
Results shown in Fig. 7 (Fig. 8). On the other hand, Dox-PCL nanocapsules (250-0 µg/ml) containing equivalent amounts of the drug (Table 1) caused normal Vero cells to show a viability percentage of 35.72-100% while cancer HepG2 cells showed a viability percentage of 11.06-100% (Fig. 9). Statistical analysis using Multiple T-test showed a high significance between normal and cancer cells at all tested concentrations of Dox, blank PCL and Dox-PCL nanocapsules (p-value < 0.001). The GI 50 (inhibitory concentration to cause growth inhibition by 50%) of free Dox and Dox-PCL nanocapsules was also studied as shown in Fig. 10. For normal Vero cells, the results indicated that GI 50 of Dox-PCL was 17.99 ± 8.62 µg/ml after 72 h as compared with free Dox that had GI 50 of 16.53 ± 1.06 µg/ ml. On the other hand, the GI 50 of both 72 h treatments with free Dox and Dox-PCL on HepG2 cells were 4.22 ± 0.04 and 2.46 ± 0.49 µg/ml, respectively, Statistical analysis showed that there was a statistical significance between free Dox and Dox-PCL nanocapsules (p-value = 0.0034).

Discussion
Dox is a chemotherapeutic drug extremely used in the treatment of many types of cancer. Its mechanism of action is based on binding to the DNA strands to inhibit its macromolecular synthesis. The disadvantages of Doxbased cancer therapy include the fact that Dox has toxic side effects on both cancer and healthy cells causing heart failure. In addition, tumor cells exhibited multidrug resistance, attributed to the presence of P-glycoprotein, which can pump Dox out, resulting in reducing its intracellular accumulation and decreasing its therapeutic efficacy. These are the major drawbacks of Dox efficiency against cancer therapy which results in restriction of its clinical applications and the need to develop new drug formulations [41]. These requirements have resulted in a trend towards miniaturization, which has challenged scientists from multidisciplinary fields to engineer novel drug delivery systems. Furthermore, several drug molecules cannot be formulated or administered by conventional techniques as they exhibit poor encapsulation efficiency of hydrophilic anticancer drugs such as Dox or suffer from limited sustained release in a complex environment such as the human body [42]. The ideal drug delivery system (DDS) should be capable of delivering a wide range of anticancer drugs for a sustained period of time into the target sites in a slow release manner. This provides an enhanced antitumor efficacy with reduced systemic side effects. Moreover, these DDS can protect drugs against their rapid metabolism and clearance by the liver, kidneys, and reticuloendothelial system, which then enhance drug stability and target specificity [22,43,44].
Due to their exceptional properties compared with bulk materials, nanoparticles applications are now found in various practical fields. In this regard, a combination of pharmacology and nanotechnology has contributed to produce more effective anticancer agents by decreasing the resistance of cancer cells. There are many types of metallic nanoparticles with several physicochemical properties such as selenium, silver, gold, titanium dioxide, zinc oxide, copper oxide, platinum, and magnesium oxide, which have been well known as anticancer carriers [45]. However, metallic drug nanocarriers result in raising issues of toxicity to humans and the environment. The insoluble metallic nanoparticles are likely to be accumulated in sensitive organs such as the heart, liver, spleen, kidney, and brain after inhalation, ingestion, and skin contact. Also, in vitro and in vivo studies had provided that the exposure to metallic nanoparticles could induce the production of reactive oxygen species (ROS), which is a predominant mechanism leading to organ toixicity [46]. Thus, efforts are focusing on using biocompatible proteins and polymers such as albumin nanoparticles [47]. But, the major obstacles associated with using albumin as DDS are; low stability, poor batch-to-batch reproducibility, difficulty in sterilization, only used with hydrophobic drugs, and low drug loading capacity. Therefore, albumin is not suitable to encapsulate the highly hydrophilic anticancer Dox.HCl drug [48]. As the selection of a suitable polymer is a crucial step, polycaprolactone (PCL) was chosen in our study to produce polymeric nanocapsules. PCL is a biodegradable and biocompatible synthetic aliphatic polyester approved by FDA. PCL and its derivatives are nontoxic and have high permeability to a lot of drugs. Properties of PCL like thermal responsiveness, drug entrapment, degradation kinetics, mechanistic properties, fabrication easiness, and controlled release nature make it a promising polymer for colloidal drug delivery applications. Most importantly, PCL is a very hydrophobic crystalline polymer, which is broken down by hydrolysis of its ester bonds under the normal physiological conditions in the human body and has minimal or no toxicity. Therefore, it has taken the attention of researchers as an applicant of choice for use in drug delivery applications and longterm implantable devices for both lipophilic and hydrophilic drugs [49]. In this study, the biocompatible and nontoxic PCL polymeric nanocapsules were successfully loaded with Dox by a modified double emulsion technique (W/O/W) according to Katata et al. [33]. The double emulsion technique was chosen to encapsulate the hydrophilic Dox.HCl inside the first (inner) aqueous phase of the core-shell nanocapsules to provide a high drug loading content (16.68 ± 2%) as well as a specific and sustained release of the anticancer drug at the cancer cells only. This leads to the reduction of its damaging side effects on normal cells, and then enhances its therapeutic applications [50]. According to [33], the hydrophilic Dox.HCl, a toxic chemotherapeutic drug model, was loaded into PCL nanocapsules using the double emulsion (W1/O/W2) method. The compatibility between Dox and PCL were approved, whereas the ability of the inner core to encapsulate Dox is largely dependent upon the compatibility between the hydrophobic polymer and the drug molecule [51]. Furthermore, our results are in line with [52,53], who selected the double emulsion technique due to its advantageous capability to encapsulate both hydrophilic and hydrophobic drugs using PCL.
For intravenous administration, nanomedicines with high drug loading content of more than 10% are favorable for cancer therapy, to reduce the drawbacks associated with nanocarrier materials having low drug content [54]. The prepared Dox-PCL nanocapsules were analyzed using DLS and were found to have a size of 212 ± 2 nm, a charge (ZP) of -22.3 ± 2 mv, and PDI of 0.019 ± 0.01 with a narrow monodispersed unimodal size distribution pattern. TEM imaging revealed that the prepared nanocapsules were spherical as shown in Fig. 2. Previous studies have shown that the shape of nanoparticles plays an important role in the delivery of the anticancer drug, especially in biological practices, including internalization by passage through the blood circulation system and directing to the sites of cancer [55]. Transport of spherical nanoparticles is expected to be much easier because of their characteristic symmetry, whereas nonspherical nanoparticles may align or tumble in the presence of flow [56]. The size of nanoparticles used in drug delivery systems should be in the range of (10-250) nm, which is large enough to prevent their rapid leakage from blood capillaries but small enough to escape capture by fixed macrophages that are lodged in the reticuloendothelial system (RES), such as the spleen and liver [22,57]. Moreover, zeta potential (ZP) is an important parameter to predict the storage stability of the nanoparticles colloidal suspension. High values of ZP, either positive or negative, are required to confirm stability and avoid aggregation of the particles by electrostatic repulsive forces [58,59]. ZP of more than ± 13 mV indicates stable nanoparticles [60]. PDI is a numerical value that represents the homogeneity of the sample. If PDI is below 0.4, the particles of the sample are considered homogenous (i.e., similar size). If PDI is higher than 0.4, the sample is less homogenous. If PDI is more than 1, the sample is completely heterogeneous [61]. In this study, a pH of 4.8 was selected to mimic the pH of cancer cells, while a pH of 7.4 was selected to mimic the pH of the healthy cells. It was observed that the Dox release from the polymeric nanocapsules was faster (~ 97% within 72 h) at pH 4.8 than at pH 7.4 (~ 97% over 23 days), which is an excellent indicator of the selectivity of Dox-PCL nanocapsules to be released at the cancer site (pH 4.8), while Dox was hardly released at the normal site (pH 7.4). According to [62], a free Dox solution was used as a positive control, and showed a relatively fast release of Dox, reaching 100% release of the total theoretical amount of Dox at 2 h and 35 min at pH 7.4. Also in the study of [63], compared with the free Dox that released to the extent of 92% within 2 h, the Doxloaded PLA-PEG-FA SPIONs showed sustained release at a steady rate of Dox by diffusing through the polymeric matrix for 120 h. In our study, the pH 4.8 was selected to mimic pH of cancer cells, while pH 7.4 was selected to mimic pH of the healthy cells. It could be observed that Dox release from Dox-PCL was faster at pH 4.8 than at pH 7.4. This was obvious also when the color of incubation media was changed after 12 h of incubation. During the releasing process, Dox was first released inside the hydrophobic core region of the polymeric nanocapsules, then diffused out from the hydrophilic outer shell of the nanocapsules, and eventually into the incubation medium. This delay of drug release indicates the nanoparticle applicability as a powerful drug carrier that minimizes the exposure of healthy tissues while increasing the accumulation of therapeutic drug in the tumor site resulting no more Dox-repeated treatment which was the main reason of cardiotoxicity.
Moreover, our results are significant to the study done by [64], which reported that in the in vitro release rate of Dox from Thermo/pH-responsive targeted polymeric nanocapsules (p(NIPA-co-AAc-co-GAA)), fabricated through double emulsion solvent evaporation technique with drug loading content of 24.31%, was much higher under acidic pH 3.0 than under physiological pH 7.4. Moreover, our results are advantageous over that from [65], in which magnetic nanoparticles were prepared based on cyclodextrin dendritic-graphene oxide as nanocarriers for Dox, with a drug loading capacity of only 9.8%, while our Dox-PCL nanocapsules showed a higher drug loading capacity reaching 16.88%. Moreover, our results are significant to [66], showing that the in vitro release rate of Dox from selenium nanoparticles (SeNPs) decorated with hyaluronic acid (HA), HA-SeNPs nanoparticles, was up to 76.9% at 30 h. Nevertheless, the release rate of Dox in PBS at pH 7.4 was about 53.5%. Our results are advantageous over that of [67], who prepared a magnetic iron oxide nanoparticles (IONPs) stabilized with trimethoxysilylpropyl-ethylenediamine triacetic acid (EDT) were developed as a nanocarrier for anticancer drug doxorubicin, with drug loading capacity of only 5 ± 0.05%, while our dox-PCL nanocapsules showed a higher drug loading capacity reaching 16.88%. Also, our results are advantageous over that from [68], in which Dox-PLGA-lecithin PEG biotin NPs were synthezised with drug loading content of only 7.21%, after transformation of hydrophilic Dox.HCl into hydrophobic Dox. The in vitro release studies showed that more than 50% of Dox were released from PLPB-NPs after 96 h of incubation at pH 7.4.
The effects of free Dox and Dox-loaded PCL nanocapsules on the viability of different cell lines, but not Vero and HepG2, were examined by the MTT assays, to evaluate their cytotoxicity and pharmaceutical efficacy. In our study, blank PCL nanocapsules have been studided in parallel and results showed that the empty vehicles were safe on normal Vero cells (97-100% viability) and had a negligible cytotoxic effect on HepG2 cells (93-100% viability). Which agrees with [69,70], approving that the blank PCL nanocapsules have almost no cytotoxic effect on the normal Vero cell line. Also, according to [71], the PCL showed in vitro antiproliferative and antioxidant effects against HepG2 cancer cells while causing opposite action on healthy hepatocytes by increasing their survival until 4 weeks in culture. Hence, the effect of empty PCL nanocapsules could be neglected in our study for both the normal Vero and cancer HepG2 cell lines. Our results from MTT test reported that in comparison to free drug, Dox-PCL nanocapsules had a more therapeutic effect against the viability of HepG2 cancer cells but a less cytotoxic effect on normal Vero cells. In which, cancer cells were significantly more sensitive to encapsulated than free drug resulting in cell viability percentages of (11.06 to 100%) and (15.31 to 100%), respectively. In contrast, the viability of normal Vero cells was significantly increased after drug encapsulation (free Dox: 34.77 to 100%; Dox-PCL: 35.72 to 100%). These differences in cytotoxicity between free Dox and Dox-loaded nanocapsules could be attributed to the different cellular uptake mechanisms of the drug, in which the cellular uptake of free Dox occurs through a passive diffusion mechanism. This is reasonable since Dox could move freely through both the plasma membrane and nuclear membrane, which may result in the trapping of the drug at the P-gap junction counteractive effects in healthy cells. However, in the case of Dox-loaded nanocapsules, the drug is released in a time-dependent manner from the nanocapsules before applying its effect on the cells, thus drug release increases with time which results in increasing the concentration and inhibiting the cell growth [72,73]. The mechanism of Dox release from the nanocapsules could be attributed to the acidic environment at the tumor cells resulting in the dissociation of Dox-loaded nanoparticles and rapid release of the Dox. As a small molecule, the uptake of free Dox is a dynamic process and it can freely escape from the cells, while the cellular uptake of the larger Dox-loaded nanocapsules is done through a non-specific endocytosis, which may lead to a reduced effect of cytosolic free Dox on the P-glycoprotein (P-gp) pumping action. Likely, the prolonged circulation and passive tumor targeting delivery process caused by the EPR effect could enhance the delivery of Dox into the tumor cells, and once the nanocapsules were internalized, it is not easy for them to escape from the cells. P-gp can recognize and eject the anticancer drug Dox from the tumor cells only when it is located in the plasma membrane, and not in the cytoplasm or lysosomes after endocytosis [74].
Our data also confirmed that after 72-h incubation, the GI 50 of Dox-PCL on normal Vero cells was higher than that of Dox used alone (Dox-PCL: 17.99 ± 8.62 µg/ ml vs free Dox:16.53 ± 1.06 µg/ml). On the other hand, a higher cytotoxic effect (i.e., lower GI 50) against the hepatic cancer line was achieved in favour of the prepared nanocapsules compared to the free Dox (Dox-PCL: 2.46 ± 0.49 µg/ml vs free Dox: 4.22 ± 0.04 µg/ml).

Conclusions
Nontoxic polymeric Dox-loaded PCL nanocapsules were successfully developed by double emulsion technique (Fig. 11) with a high drug loading content of 16.18 ± 2%. A stable colloidal suspension of spherical Dox-PCL nanocapsules (particle size: 212 ± 2 nm; ZP: -22.3 ± 2 mv; PDI: 0.019 ± 0.01) with a narrow size distribution pattern was characterized. MTT test and GI 50 results confirmed that in comparison to equivalent amounts of free Dox, the prepared encapsulated drug exhibited a higher in vitro therapeutic cytotoxicity on cancer HepG2 cell line, while being less toxic on normal Vero cells. The Dox was released from Dox-PCL nanocapsules at pH 4.8 (cancer pH) faster than at pH 7.4 (healthy pH); 98% and 26 over 72 h, respectively. Finally, the polymeric based drug delivery system offers a successful and promising potential application for many therapeutic agents with more confidence in Dox for the clinical treatment of hepatocellular carcinoma.

Recommendations
Further in vivo studies as well as fabrication of Dox-PCL nanocapsules with both targeting (folic acid) and labeling (rhodamine) agents would be of useful importance in evaluating the potency and pharmacokinetics of these promising biocompatible Dox nanocarriers.